The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.
Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, megalomicin, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; WO 97/02358; and WO 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or xe2x80x9cmodularxe2x80x9d and Type II xe2x80x9citerativexe2x80x9d PKS enzymes.
Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, megalomicin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Each ORF of a modular PKS can comprise one, two, or more xe2x80x9cmodulesxe2x80x9d of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or xe2x80x9cdomains.xe2x80x9d These large multifunctional enzymes ( greater than 300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying xcex2-carbon processing activities (see O""Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a xe2x80x9ccleanxe2x80x9d host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of xcex2-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
Megalomicin is a macrolide antibiotic produced by Micromonospora megalomicea, a member of the Actinomycetales family of soil bacteria that produces many types of biologically active compounds. Megalomicin is a glycoside of erythromycin A, a widely used antibacterial drug with little or no antimalarial activity. Megalomicin has antibacterial properties similar to those of erythromycin, and in 1998, it was discovered also to have potent antiparasitic activity and low toxicity. The antiparasitic activity may be related to the effect megalomicin has on protein trafficking in eukaryotes, where it appears to inhibit vesicular transport between the medial and trans-Golgi, resulting in under-sialylation of proteins. Hence, megalomicin offers an exciting opportunity to develop a new class of antiparasitic drugs with a different mechanism of action than the drugs currently in use and, therefore, possibly active against drug-resistant forms of Plasmodium falciparum. 
The number and diversity of megalomicin derivatives have been limited due to the inability to manipulate the PKS genes, which have not previously been available in recombinant form. Genetic systems that allow rapid engineering of the megalomicin biosynthetic genes would be valuable for creating novel compounds for pharmaceutical. agricultural, and veterinary applications. The production of such compounds could be more readily accomplished if the heterologous expression of the megalomicin biosynthetic genes in Streptomyces coelicolor and S. lividans and other host cells were possible. The present invention meets these and other needs.
The present invention provides recombinant methods and materials for expressing PKS enzymes and polyketide modification enzymes derived in whole and in part from the megalomicin biosynthetic genes in recombinant host cells. The invention also provides the polyketides produced by such PKS enzymes. The invention provides in recombinant form all of the genes for the proteins that constitute the complete PKS that ultimately results, in Micromonospora megalomicea, in the production of megalomicin. Thus, in one embodiment, the invention is directed to recombinant materials comprising nucleic acids with nucleotide sequences encoding at least one domain, module, or protein encoded by a megalomicin PKS gene. In one preferred embodiment of the invention, the DNA compounds of the invention comprise a coding sequence for at least one and preferably two or more of the domains of the loading module and extender modules 1 through 6, inclusive, of the megalomicin PKS.
In one embodiment, the invention provides a recombinant expression vector that comprises a heterologous promoter positioned to drive expression of one or more of the megalomicin biosynthetic genes. In a preferred embodiment, the promoter is derived from another PKS gene. In a related embodiment, the invention provides recombinant host cells comprising one or more expression vectors that produce(s) megalomicin or a megalomicin derivative or precursor. In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor. 
In another embodiment, the invention provides a recombinant expression vector that comprises a promoter positioned to drive expression of a hybrid PKS comprising all or part of the megalomicin PKS and at least a part of a second PKS. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the hybrid PKS and its corresponding polyketide. In a preferred embodiment, he host cell is Streptomyces lividans or S. coelicolor. 
In a related embodiment, the invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by hybrid PKS enzymes of the invention. The resulting polyketides can be further modified to convert them to other useful compounds, such as antibiotics, motilides, and antiparasitics, typically through hydroxylation and/or glycosylation. Modified macrolides provided by the invention that are useful intermediates in the preparation of antiparasitics are of particular benefit.
In another related embodiment, the invention provides a method to prepare a nucleic acid that encodes a modified PKS, which method comprises using the megalomicin PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, deletion, insertion, or replacement. The thus modified megalomicin PKS encoding nucleotide sequence can then be expressed in a suitable host cell and the cell employed to produce a polyketide different from that produced by the megalomicin PKS. In addition, portions of the megalomicin PKS coding sequence can be inserted into other PKS coding sequences to modify the products thereof.
In another related embodiment, the invention is directed to a multiplicity of cell colonies, constituting a library of colonies, wherein each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the megalomicin PKS. Thus, at least a portion of the modular PKS is identical to that found in the PKS that produces megalomicin and is identifiable as such. The derived portion can be prepared synthetically or directly from DNA derived from organisms that produce megalomicin. In addition, the invention provides methods to screen the resulting polyketide and antibiotic libraries.
The invention also provides novel polyketides, motilides, antibiotics, antiparasitics and other useful compounds derived therefrom. The compounds of the invention can also be used in the manufacture of another compound. In a preferred embodiment, the compounds of the invention are formulated in a mixture or solution for administration to an animal or human.
In a specific embodiment, the invention provides an isolated nucleic acid fragment comprising a nucleotide sequence encoding a domain of megalomicin polyketide synthase (PKS) or a megalomicin modification enzyme. The isolated nucleic acid fragment can be a DNA or a RNA. Preferably, the isolated nucleic acid fragment is a recombinant DNA compound.
The isolated nucleic acid fragment can comprise a single, multiple or all the open reading frame(s) (ORF) of the megalomicin PKS or a megalomicin modification enzyme. Exemplary ORFs of megalomicin PKS include the ORFs of the megAI, megAII and megAIlI genes. The isolated nucleic acid fragment can also encode a single, multiple, or all of the domains of the megalomicin PKS. Exemplary domains of the megalomicin PKS include a TE domain, a KS domain, an AT domain, an ACP domain, a KR domain, a DH domain and an ER domain. In a preferred embodiment, the nucleic acid fragment encodes a module of the megalomicin PKS. In another preferred embodiment, the nucleic acid fragment encodes the loading module, a thioesterase domain, and all six extender modules of the megalomicin PKS.
Megalomicin modification enzymes include those enzymes involved in the conversion of 6-dEB into a megalomicin such as the enzymes encoded by the megE, meg BV, megCIII, megK, megDI and megG (renamed megY) genes. Megalomicin modification enzymes also include those enzymes involved in the biosynthesis of mycarose, megosamine or desosamine, which are used as biosynthetic intermediates in the biosynthesis of various megalomicin species and other related polyketides. The enzymes that are involved in biosynthesis of mycarose, megosamine or desosamine are described in FIGS. 5 and 10.
In a preferred embodiment, the invention provides an isolated nucleic acid fragment which hybridizes to a nucleic acid having a nucleotide sequence set forth in the SEQ. ID NO:1, under low, medium or high stringency. More preferably, the nucleic acid fragment comprises, consists or consists essentially of a nucleic acid having a nucleotide sequence set forth in the SEQ. ID NO:1.
In another specific embodiment, the invention provides a substantially purified polypeptide, which is encoded by a nucleic acid fragment comprising a nucleotide sequence encoding a domain of megalomicin polyketide synthase (PKS) or a megalomicin modification enzyme. The polypeptide can comprise a single domain, multiple domains or a full-length megalomicin PKS or megalomicin modification enzyme. Functional fragments, analogs or derivatives of the megalomicin PKS or megalomicin modification enzyme polypeptides are also provided. Preferably, such fragments, analogs or derivatives can be recognized by an antibody raised against a megalomicin PKS or megalomicin modification enzyme. Also preferably, such fragments, analogs or derivatives comprise an amino acid sequence that has at least 60% identity, more preferably at least 90% identity, to their wild type counterparts.
In still another specific embodiment, the invention provides an antibody, or a fragment or derivative thereof, which immuno-specifically binds to a domain of megalomicin polyketide synthase (PKS) or a megalomicin modification enzyme. The antibody can be a monoclonal or polyclonal antibody or an antibody fragment. Preferably, the antibody is a monoclonal antibody.
In yet another specific embodiment, the invention provides a recombinant DNA expression vector comprising the recombinant DNA compound encoding at least a domain of the megalomicin PKS or a megalomicin modification enzyme, wherein said domain is operably linked to a promoter. Preferably, the recombinant DNA expression vector further comprises an origin of replication or a segment of DNA that enables chromosomal integration.
In yet another specific embodiment, the invention provides a recombinant host cell comprising the above-described recombinant DNA expression vector encoding at least a domain of megalomicin PKS or the megalomicin modification enzyme. The recombinant host cells can be any suitable host cells including animal, mammalian, plant, fungal, yeast, and bacterial cells. Preferably, the recombinant host cells are Streptomyces cells, such as Streptomyces lividans and S. coelicolor cells, or ccharopolyspora cells, such as Saccharopolyspora erythraea cells. Also preferably, the recombinant host cells do not produce megalomicin in their untransformed, non-recombinant state.
When the recombinant host cell contains nucleic acid encoding more than one megalomicin PKS or megalomicin modification enzyme, or domains thereof, such nucleic acid material can be located at a single genetic locus, e.g., on a single plasmid or at a single chromosomal locus, or at different genetic loci, e.g., on separate plasmids and/or chromosomal loci. In one example, the invention provides a recombinant host cell, which comprises at least two separate autonomously replicating recombinant DNA expression vectors, and each of said vectors comprises a recombinant DNA compound encoding a megalomicin PKS domain or a megalomicin modification enzyme operably linked to a promoter. In another example, the invention provides a recombinant host cell, which comprises at least one autonomously replicating recombinant DNA expression vector and at least one modified chromosome, each of said vector(s) and each of said modified chromosome comprises a recombinant DNA compound encoding a megalomicin PKS domain or a megalomicin modification enzyme operably linked to a promoter. Preferably, the autonomously replicating recombinant DNA expression vector and/or the modified chromosome further comprises distinct selectable markers.
In a preferred embodiment, the cell comprises three different vectors, one of which is integrated into the chromosome and two of which are autonomously replicating, and each of the vectors comprises a meg PKS gene. Optionally, one or more of the meg PKS genes contains one or more domain alterations, such as a deletion or substitution of a meg PKS domain with a domain from another PKS.
In yet another specific embodiment, the invention provides a hybrid PKS, which is produced from a recombinant gene that comprises at least a portion of a megalomicin PKS gene and at least a portion of a second PKS gene for a polyketide other than megalomicin. For example, and without limitation, the second PKS gene can be a narbonolide PKS gene, an oleandolide PKS gene, or a rapamycin PKS gene. In one embodiment, the hybrid PKS is composed of a loading module and six extender modules, wherein at least one domain of any one of extender modules 1 through 6, inclusive, is a domain of an extender module of megalomicin PKS. In another preferred embodiment, the hybrid PKS comprises a megalomicin PKS that has a non-functional KS domain in module1.
In yet another specific embodiment, the invention provides a method of producing a polyketide, which method comprises growing the recombinant host cell comprising a recombinant DNA expression vector encoding at least a domain of the megalomicin PKS or a megalomicin modification enzyme under conditions whereby the megalomicin PKS domain or the megalomicin modification enzyme comprised by the recombinant expression vector is produced and the polyketide is synthesized by the cell, and recovering the synthesized polyketide. Preferably, the recombinant host cell comprises a recombinant expression vector that encodes at least a portion of a megAI, megAII, or megAIII gene.
These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.